Under conditions of reduced oxygen tension (hypoxia) production of tissue vascular endothelial growth factor (VEGF) increases, resulting in neovascularization of. i) arthritic joints in rheumatoid arthritis, ii) atherosclerotic plaques, or iii) tumors. In our model of post-transcriptional regulation, VEGF expression is controlled either by increases in mRNA stability or by release from translational repression. The factors, in our model, that mediate increased VEGF production are: i) trans-acting RNA binding proteins that act on, ii) cis AU-rich response elements (AURE) in the VEGF of VEGF mRNA. We propose to characterize the role o f AURE in the VEGF 31UTR by performing targeted disruption of the AURE regulatory domain in the VEGF locus of the mouse. Using this approach we will produce both cultured cell lines and mice in which production of VEGF is segregated from mechanisms of post-transcriptional regulation. The Specific Aims in this proposal will evaluate the contribution of the VEGF AURE domain as follows: In Aim I we will produce a targeting vector for homologous recombination and use it to disrupt the AURE domain in the VEGF of VEGF in mouse embryonic stem (ES) cells. This disruption will not affect the coding region of the VEGF locus, but because AURE are often de-stabilizing, we suspect that VEGF expression may be up-regulated. In Aim 2 we will determine if the targeted disruption of the VEGF AURE produces phenotypic effects on: i) VEGF production, or ii) the tumorigenicity of ES cells in nude mice. In Aim 3 we will ask if the phenotypic changes measured in VEGF AURE mutants result from changes in the turnover or translational repression of VEGF mRNA. Finally, in Aim 4, we will use the VEGF AURE mutant cells to create homozygous and heterozygous mouse strains and determine the effects of VEGF overproduction on embryogenesis and on the physiology and pathophysiology of adult mice. The VEGF AURE mutant cell lines developed in this proposal will be invaluable in studies of embryogenesis and, if VEGF AURE mutant mice are produced, these mice will provide an exciting system with which to study overvascularization, as is found in arthritic joints, tumors and proliferative retinopathy, and to study the effects of VEGF on immune cells, including monocytes and osteoclasts in arthritic disease.